Vigna Unguiculata (L.) Walp.]

Plant Reprod (2016) 29:165–177 DOI 10.1007/s00497-015-0273-3

ORIGINAL ARTICLE

New observations on gametogenic development and reproductive experimental tools to support seed yield improvement in cowpea [Vigna unguiculata (L.) Walp.]

1 2 1 Rigel Salinas-Gamboa • Susan D. Johnson • Nidia Sa´nchez-Leo´n • 2 1 Anna M. G. Koltunow • Jean-Philippe Vielle-Calzada

Received: 28 October 2015 / Accepted: 21 December 2015 / Published online: 4 January 2016 Ó The Author(s) 2016. This article is published with open access at Springerlink.com

Key message Cowpea reproductive tools. ﬂoral bud size and reproductive organ development, Abstract Vigna unguiculata L. Walp. (cowpea) is recog- determining that gametogenesis occurs more rapidly in the nized as a major legume food crop in Africa, but seed anther than in the ovule. We also show that the mode of yields remain low in most varieties adapted to local con- megagametogenesis is of the Polygonum-type and not ditions. The development of hybrid cowpea seed that could Oenothera-type, as previously reported. Finally, we be saved after each generation, enabling signiﬁcant yield developed a whole-mount immunolocalization protocol increases, will require manipulation of reproductive and applied it to detect meiotic proteins in the cowpea development from a sexual to an asexual mode. To develop megaspore mother cell, opening opportunities for com- new technologies that could support the biotechnological paring the dynamics of protein localization during male manipulation of reproductive development in cowpea, we and female meiosis, as well as other reproductive events in examined gametogenesis and seed formation in two this emerging legume model system. transformable, African-adapted, day-length-insensitive varieties. Here, we show that these two varieties exhibit Keywords Megagametogenesis Á Megasporogenesis Á distinct morphological and phenological traits but share a Microgametogenesis Á Cowpea Á Reproductive calendar Á common developmental sequence in terms of ovule for- Whole-mount immunolocalization Á Ovule mation and gametogenesis. We present a reproductive calendar that allows prediction of male and female gametogenesis on the basis of sporophytic parameters related to Introduction

Many of the poorest countries in the world derive 10–20 % Communicated by Dolf Weijers. of their total dietary protein from grain legumes, members of the Fabaceae (Akibode and Maredia, 2011). Cowpea, Anna M. G. Koltunow and Jean-Philippe Vielle-Calzada are co-senior Vigna unguiculata (L.) Walp. is one of the eight grain authors. legumes currently being targeted for yield and agronomic Electronic supplementary material The online version of this improvement by the Consultative Group for International article (doi:10.1007/s00497-015-0273-3) contains supplementary Agricultural Research (CGIAR). Cowpea originated in material, which is available to authorized users. Africa, which produces over 95 % of the 5.4 million tons & Jean-Philippe Vielle-Calzada of dried cowpeas produced globally, on an estimated 11.5 [email protected] million hectares of land across a diverse range of farming

1 systems, in the semiarid regions of West and Central Grupo de Desarrollo Reproductivo y Apomixis, Laboratorio Africa. Approximately 200 million individuals are esti- Nacional de Geno´mica para la Biodiversidad, CINVESTAV Irapuato, CP 36821 Guanajuato, Mexico mated to consume the grain daily in West Africa as it provides a high source of protein (25 % by weight) and all 2 Agriculture Flagship, Commonwealth Scientific and Industrial Research Organization, plant organs are edible. Cowpea is an important component Private Bag 2, Glen Osmond, SA 5064, Australia in increasing sustainable farming production by being 123 166 Plant Reprod (2016) 29:165–177 drought resistant and having the ability to fix atmospheric megagametogenesis in Vigna. An important prerequisite nitrogen, contributing to reduced soil erosion. for developing robust hybrid seed production systems or Conventional cowpea breeding is currently underpinned attempting to alter reproduction in cowpea is a clear by the use of molecular markers (Lucas et al. 2011, 2013), understanding of the temporal and spatial events of sexual correlations between genetic and physical maps (Pottorff male and female gametophyte formation in relation to et al. 2014), and large-scale genomic sequencing (Andargie flower morphology and supporting cytological tools to et al. 2011; Barrera-Figueroa et al. 2011; Huang et al. efficiently and accurately examine changes induced by 2012). Transformation technologies have been established hybridization, mutation, or transgenic approaches. for cowpea and are being implemented to accelerate In this paper, we applied a range of cytological techniques agronomic performance, with strong indications of success to a pair of cultivated African cowpea varieties of known in the development of seed borer-resistant lines (Kang et al. pedigree developed by the International Institute of Tropical 2014; Popelka et al. 2006). Despite advances in increasing Agriculture (Kang et al. 2014) and developed a reproductive cowpea yields and stress tolerance over the last two dec- calendar that allows prediction of male and female game- ades, many varieties are still low yielding and susceptible togenesis on the basis of sporophytic parameters such as to a range of biotic and abiotic stresses (Lush and Evans floral bud size, gynoecium length, and integument growth. 1981; Singh 2014; Huynh et al. 2015; Lucas et al. 2015). Although one flowers earlier than the other, both share a The development of cowpea hybrids has the potential to common developmental sequence in terms of ovule forma- increase crop yields by 30 % as suggested by published tion and gametogenesis. Contrary to previous reports, we studies aimed at identifying levels of heterosis in progeny show that the female gametophyte is not of the Oenothera - from crosses between inbred cowpea parents (Bhaskaraiah type but consistently develops from three mitotic nuclear et al. 1980; Ortiz 1998; Ushakumari et al. 2010; Kumari divisions of the most chalazal megaspore, giving rise to a et al. 2012). Hybrid seed production systems have not yet megagametophyte of the Polygonum-type. Callose staining been developed in cowpea. Impediments include self-fer- of pollen tubes and tracking of sperm cells within the tilizing hermaphrodite flowers, low outcrossing rates megagametophyte showed that fertilization occurs 12–14 h (\5 %; Timko and Singh 2008), and high flower drop rates after pollination. We also developed a simple whole-mount (Wien and Summerfield 1980; Ehlers and Hall 1996; immunolocalization procedure with sufficient sensitivity to Ojehomon and Samyaolu 1970). In all current hybrid seed allow the detection of meiotic proteins specifically expressed production systems, hybrid seeds need to be generated and in the megaspore mother cell. Collectively, the methods purchased each growing season, impeding their purchase developed here for cowpea are likely to be applicable to by poor farmers. The current inability to produce hybrid analyses of gametogenesis in a range of legumes. seeds relates to the plant’s sexual reproductive process. Meiosis and recombination during gamete formation and gamete fusion at fertilization alleviate heterosis, concur- Materials and methods rently leading to trait segregation. Development of cowpea hybrids from which seeds could be saved by farmers would Plant material and growth conditions offer the potential to generate higher-yielding cowpea crops in an economical manner. The generation of such Seeds of Vigna unguiculata IT97K-499-35 and IT86D- ‘‘self-reproducing’’ hybrids would require changing 1010 were germinated and grown in either a soil mixture of reproduction in the sexually reproducing hybrid to an 3:1:1 sunshine soil/vermiculite/perlite (v/v/v), and Osmo- asexual seed forming mode mimicking the events of apo- cote (1.84 kg/m3) as a fertilizer, or a mixture of BioGrow mixis to generate clonal seed where vigor was preserved. (60 % coarse milled composted pine bark[7 mm, 30 % of Recent studies have provided proof-of-concept of clonal fine milled composted pine bark 3–6 mm, and 10 % of seed generation, which might be applicable for the gener- washed coarse river sand) and Osmocote (1.84 kg/m3). ation of self-reproducing cowpea hybrids (Marimuthu et al. Both varieties were grown under greenhouse condi- 2011; Hand and Koltunow 2014; Gilbert 2015). tions but did not flower during winter; temperatures in While general descriptions of reproductive development Irapuato were 25–28 °C (spring–summer) and 20–24 °C in members of the Fabaceae have been published from a (autumn–winter), and temperatures in Adelaide were physiological and developmental perspective (Brown 1917; 22–28 °C (summer) and 18–23 °C (winter). Mitchel 1975; Rembert 1977; Albertsen and Palmer 1979; Kennell and Horner 1985; Moço and Mariath 2003; Cytological analysis Soverna et al. 2003; Rodriguez-Rianõs et al. 2006; Chehregani and Tanaomi 2010; Ghassempour et al. 2011), For light microscopy analysis, floral buds were manually there is surprisingly scarce information on micro- and dissected and fixed in FAA (ethanol (50 %), formaldehyde 123 Plant Reprod (2016) 29:165–177 167

(10 %), acetic acid (4 %), and water) for at least 12 h, Whole-mount protein immunolocalization before being dissected with hypodermic needle and scalpel. Both the bud and the gynoecia length were measured Immunolocalization was performed as previously descri- before proceeding to dehydration. Individual gynoecia bed (Escobar-Guzman et al. 2015), with modifications. were dissected under a stereoscope to expose the ovules Developing ovules were fixed in paraformaldehyde without detaching them from the placenta. Strings of (1 9 PBS, 4 % paraformaldehyde, 2 % Triton), under ovules were progressively dehydrated in ethanol gradients: continuous agitation for 2 h on ice, washed three times in 15, 25, 40, 50, 60, 75, and 100 % for 30 min each, then a 1 9 PBS, and embedded in 15 % acrylamide/bisacry- ethanol/methyl salicylate gradient 2:1, 1:1, and 1:2 for lamide (29:1) on pre-charged slides (Fisher Probe-On) 20 min each, and finally mounted on regular microscope treated with poly-L-Lysine as described (Bass et al. 1997). slides and 3 coverslips: two on each side of the slide and Gynoecia were gently opened to expose ovules by pressing the third one completely covering the tissue mounted with a coverslip on top of the acrylamide. Samples were methyl salicylate 100 %, or directly on well slides. Sam- digested in an enzymatic solution composed of 1 % ples were observed under Nomarski illumination using a driselase, 0.5 % cellulase, 1 % pectolyase (all from Sigma) DMR Leica or Zeiss microscope. For cytological analysis in 1 9 PBS for 80 min at 37 °C, subsequently rinsed three under confocal microscopy, samples were fixed in FPA times in 1 9 PBS, and permeabilized for 2 h in 1X PBS/ (formalin, propionic acid, and ethanol 10:5:7) for at least 2 % Triton. Blocking was 1 % BSA (Roche) for 1 h at 12 h, rinsed in L-arginine (12.4 pH), incubated in RNAse 37 °C. Slides were then incubated overnight at 4 °C with for 30 min at 37°, and stained with 0.1 M propidium iodide ASY1 primary antibody used at a dilution of 1:100 (pH 12.4) for 1.5 h on ice and in constant agitation. All (Olmedo-Monfil et al. 2010). Slides were washed for 6 h in samples were mounted on conventional microscope slides 1 9 PBS/0.2 % Triton, with refreshing of the solution with ProLong or Vectaschield (Vector Laboratories, Bur- every 2 h. The samples were then coated overnight at 4 °C lingame USA) and observed on a Zeiss LSM510 META with secondary antibody Alexa Fluor 488 (Molecular confocal laser scanning microscope with single-track con- Probes) at a concentration of 1:300. After washing in figuration and excitation with a diode-pumped solid-state 1 9 PBS/0.2 % Triton for at least 8 h, the slides were laser at 568 nm; emission was collected using a band-pass incubated with propidium iodide (PI; 500 lgmL-1)in of 575–615. 1 9 PBS for 20 min, washed for 30 min in 1 9 PBS, and mounted in PROLONG medium (Molecular Probes) overnight at 4 °C. Serial sections on Stage 1 ovules were Morphometric analysis of the floral bud, gynoecium, captured on a laser scanning confocal microscope (Zeiss and anther LSM 510 META), with multitrack configuration for detecting PI (excitation with DPSS laser at 568 nm, For flower bud size, bracts were removed and the maxi- emission collected using BP: 575–615 nm) and Alexa 488 mum length of the proximal–distal axis was used as a (excitation with Argon laser at 488, emission collected reference for length. The gynoecium was measured fol- using BP: 500–550 nm). Laser intensity and gain were set lowing the same axis, from the base of the carpel to the at similar levels for all experiments. Projections of selected apex of the style. The maximum length of the pre-meiotic optical sections were generated using ImageJ. primordium was measured from the middle part of the base, where a small protrusion is visible in the placenta, to the apex. The maximum width was taken as the distance Results across the primordium following the dorsal–ventral axis. The megaspore mother cell (MMC) was measured at its Growth, flowering time, and phenology of two maximal length following both the proximal–distal and cowpea varieties dorsal–ventral axis. After meiosis (Stage 4), the maximum length of the meiotic configuration was measured follow- Two transformable V. unguiculata varieties, IT86D-1010 ing the proximal–distal axis. The length of the anther was and IT97K-499-35 (abbreviated IT86D and IT97K, measured from its base to the most distal tip. A total data respectively) with different genetic pedigrees (Supple- matrix was generated for all four estimated parameters mentary Figure 1) were utilized in this study. Both are (gynoecium length, number of dorsal integumentary cells, photoperiod insensitive, adapted to dry, semiarid regions, length of the meiotic configuration, and width of the mei- and were developed in West Africa. In general, both otic configuration), and Pearson correlations were calcu- varieties behave similarly at both growing locations in lated using XLstat. Irapuato, Mexico, and in Adelaide, Australia, stably

123 168 Plant Reprod (2016) 29:165–177

123 Plant Reprod (2016) 29:165–177 169 b Fig. 1 Phenology and reproductive timeline in two varieties of shown in closed (Fig. 1f) and dissected floral buds cowpea. Vertically aligned micrographs in g–u represent correspond- (Fig. 1g–k). In both varieties, seed yields are maximal ing male and female developmental stages within the same floral bud. a Vegetative architecture and leaf morphology of IT97K. b Vegetative during this phase, with approximately 16–17 seeds setting architecture and leaf morphology of IT86D. c Mature flower of per pod. In the following 6 weeks (IT86D) to 8 weeks IT86D; the inset shows floral buds of IT97K. d Mature seeds of (IT97K), the new leaves are significantly reduced in size IT86D. e Mature seeds of IT97K. f Floral development in IT86D; the and floral abortion increases with few pods evident, har- numerical register of floral buds corresponds to developmental stages illustrated in g–u. g Dissected floral bud of 2.5 mm in length. boring an average of ten seeds. The third phase is char- h Dissected floral bud of 3 mm in length. i Dissected floral bud of acterized by cessation of leaf and flower formation and 4 mm in length. j Isolated gynoecia of 3.4 mm in length. k Dissected seed set, as the plant undergoes senescence. floral bud of 32 mm in length. l Differentiated microspore mother Male and female gametophyte development begins in cells. m A tetrad of microspores. n One-nucleate microspore. o Two- nucleate microspore. p Fully differentiated pollen grain. q Ovule developing anthers and ovules of 2.5 mm floral buds in primordium containing an archespore. r Megaspore mother cell. which the anther has reached 0.1 mm and the gynoecium s Megaspore mother cell at the onset of meiosis. t Functional and 1.5 mm in length (Fig. 1f–g). All events of pollen and degenerated megaspores. u Fully differentiated female gametophyte. female gametophyte development, progressing from pollen Arc archespore, CC central cell, DMg degenerating megaspore; EC egg cell, FM functional megaspore, M microspore, MMC megaspore mother cell formation to pollen maturity, and from arch- mother cell, PMC pollen mother cell, PN polar nucleus, SC sperm espore differentiation to full cellularization of the cell, Sy synergid, VN vegetative nucleus. Scale bars a and b = 13 cm megagametophyte, respectively, can be correlated with (inset = 4 cm); c = 5mm (inset = 0.5 mm); d, e = 4 mm; floral bud size and gynoecium length in dissected buds as f = 8.5 mm; g, h = 0.6 mm; i = 1.2 mm; j = 0.5 mm; k = 2.7 mm; l = 13 lm; m–o = 10 lm; p = 20 lm; q– shown in Fig. 1g–u and Table 1. The elongating stamens s = 10 lm; t = 11.5 lm; u = 18 lm grow and curl around the stigma of the gynoecium and dehisce, enabling fertilization when the petals are still closed (Fig. 1k), which partially explains the low degree of expressing clear inter-variety differences in leaf phenology outcrossing. (Fig. 1a, b and insets; see also ‘‘Methods’’). Contrary to Adelaide, where both varieties had a vine-like habit, in Timeline of male and female gametophytic Irapuato, IT86D presented a more vine-like habit with development numerous polychotomic ramifications and thin stems, whereas IT97K had dichotomic branching (Fig. 1a, b). Early ovule development Although petals are white with subtle purple during floral development in both IT86D and IT97K, the flowers Analyses of the first phase of flowering in both IT86D and become yellow at the onset of senescence (Fig. 1c). During IT97K varieties showed that the ovules initiate as elon- the first five (IT86D) to eight (IT97K) weeks after flow- gated primordia, emerging anticlinally from internal cell ering, a variable proportion (50–70 %) of flowers aborted layers of an incipient gynoecium that is approximately per plant at both locations during the life cycle of both 0.5 mm in length from its base to its apex. When the ovule varieties, indicating a variability in the number of flowers primordium reaches 20–50 lm in length, the L2 and L3 that give rise to seeds. At 40–48 h following anthesis and layers exhibit a cluster of differentiated cells having an fertilization, the corolla is usually fully detached from the enlarged nucleus and nucleolus (Fig. 2a, b and Table 1), peduncle and only the calyx remains attached at the onset indicative of active cell division. The first stages of pri- of fruit formation. Non-abortive flowers give rise to pods mordium elongation, which occur prior to integumentary harboring between 8 and 17 seeds per pod. Seeds of IT86D initiation, are characterized by a rapid phase of sporophytic can be distinguished from those of IT97K by additional ovule cell proliferation. During these stages, the gynoecia purple pigmentation surrounding the hilum or ‘‘black-eye’’ grow from 0.5 to 1.5 mm (Figs. 1g, q and 2c; Table 1). of the seed in IT86D (Fig. 1d, e). Importantly, flowering time differs in both varieties with Differentiation and division of the megaspore mother cell flower buds evident in IT86D at 3–4 weeks (Irapuato, Mexico) or 5–6 weeks (Adelaide, Australia) following When the gynoecium and the ovule reach 1.5 mm and germination (Fig. 1c), whereas flowering is delayed until 80–160 lm, respectively, the archespore differentiates at 7–8 weeks after germination in IT97K (Fig. 1c, inset). The the distal pole of the primordium. The archespore does not life cycle of both cowpea varieties is broadly divided into abut cells in the L1 epidermal layer, but it is surrounded by three distinct growth and senescence phases. The first subepidermal and possibly L3 cell layers (Fig. 2c). At this 5 weeks (IT86D) to 8 weeks (IT97K) after germination are same stage, the anther is close to 100 lm in length and characterized by robust vegetative growth, with floral contains differentiated pollen mother cells (Figs. 1h, m; development progressing through the characteristic stages Table 1). Apart from elongation, there were no obvious 123 170 Plant Reprod (2016) 29:165–177

Table 1 Reproductive timeline: average ﬂoral bud size, average gynoecium length, and gametogenesis in cowpea Average ﬂoral bud Average gynoecium Ovule development; female and male gametogenesis size (mm) length (mm)

1 0.5 Ovule primordium initiation; active cell divisions 2.5 1.5 Rapid growth of ovule primordium (80–160 lm in length). Differentiation of subepidermal archespore; differentiation of pollen mother cells in the young anther 3 1.75 Ovule primordium reached 90–210 lm in length; differentiation of the MMC (at least 20 lmin length) 4 2.5 Differentiated MMC has entered prophase I: frequent leptotene and zygotene stages. Initial growth of inner and outer integuments. Ovule has reached a 90° angle with respect to its initial proximal–distal axis. Anther is approximately 200 lm in length; pollen development at the tetrad stage 5 3 Maximum length of MMC is reached (approximately 50 lm). Outer integument is now longer than the inner integument that covers approximately half of the nucellus. Anther approximately 280 lm in length and contains free one-nucleate microspores 5 3.5 Meiosis II has initiated and sometimes has already occurred as four meiotically derived cells are visible. Inner integument covering about half of the nucellus, outer integument has reached the micropylar pole. Rare presence of a two-nucleate stage of female gametophyte 5.5 4 Full differentiation of the functional megaspore, full degeneration of three additional megaspores. Both integuments have reached their full length. Anther is approximately 350 lm in length and contains two-nuclear microspores 5.5–6 4 Two- to four-nucleate stage of female gametophyte development. Three-nucleate pollen often visible 6.5 5–6 Four- to eight-nucleate stage of female gametophyte development. Mature pollen grain 10 7.5–8 Cellularized female gametophyte; sporadic visual identiﬁcation of rapidly degenerating antipodal cells

structural differences enabling distinction between the functional and surviving megaspore is not of micropylar archespore and the MMC, and we therefore arbitrarily location as is characteristic of developing Oenothera-type classified a cell as an MMC when the archespore enlarged embryo sacs, but is located at the most chalazal pole of the to 25 lm in length. At this time of MMC differentiation, post-meiotic tetrad. Furthermore, it undergoes three rounds the gynoecium had usually reached 2.5 mm in length and of mitosis, indicating that cowpea undergoes the classic the anther was 200 lm long and contained meiotic tetrads Polygonum-type of megagametogenesis (Fig. 2g). In 4 mm (Figs. 1h, m, r, 2d; Table 1). In the ovule, integumentary long gynoecia, megasporogenesis usually has concluded growth initiates at the dorsal side of the ovule primordium and the functional megaspore is differentiated (Fig. 1j, t). (Fig. 2d–e). Integumentary growth occurs at stages when The anthers are close to 350 lm in length at this stage, and the gynoecium length is between 2.5 and 4 mm (Fig. 1h–j). microspores have divided, characteristic of the two-nuclear The maximum length of the MMC (approximately stage (Fig. 1j, o). The outer integument has largely com- 50 lm) is usually reached in floral buds containing pleted formation of the micropyle (Fig. 2g, h), and a vas- gynoecia that are 3 mm in length (Figs. 1i, s, 2f), and cular bundle has differentiated in the funiculus, terminating when 200 lm long anthers contain uninucleate micro- in the chalazal region of the ovule. spores (Fig. 1i, n). In the ovule, the inner integument has surrounded about half of the nucellus and the outer Megagametogenesis integument is still shorter than the inner integument (Fig. 2f). When the gynoecium is approximately 3.5 mm Megagametogenesis occurs rapidly after the functional in length, the MMC undergoes or finishes meiosis II, as megaspore is around 80 lm in length along its longitudinal four meiotically derived cells are visible (Fig. 2g). The axis (Fig. 2h). Once the gynoecium is 5 mm in length, the inner integument arrests growth at this stage and does not female gametophyte is generally at the two- to four-nuclear surround the base of the embryo sac or contribute to the (2NFG and 4NFG) stage (Fig. 1k, u), and the anthers micropyle (Fig. 2h). contain tricellular pollen grains (1K and 1P). The female Meiosis occurs in the same cellular plane, giving rise to gametophyte undergoes cellularization rapidly after eight a tetrad of megaspores. Contrary to previous reports, the nuclei form in the gynoecia between 5 and 8 mm in length

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Fig. 2 Ovule and female gametophyte development in cowpea. megaspores (gynoecium length 3.5 mm). h Developing ovule at the a Initial stages of ovule primordium growth; several proliferative cells onset of megagametogenesis; a fully differentiated megaspore and differentiate in the L2 and L3 layers (arrows); the gynoecium is degenerated megaspores are within the same cellular plane (gynoe- approximately 0.5 mm in length. b Ovule primordium prior to cium length 3.75 mm). i Developing ovule at the four-nuclear stage of archespore differentiation (gynoecium 1 mm). c Developing ovules megagametogenesis. j Differentiating female gametophyte showing harboring a single archespore (gynoecium 1.5 mm). d Developing three antipodal cells at the chalazal end (inset). k Fully differentiated ovule showing an MMC at the onset of megasporogenesis (gynoe- ovule containing a cellularized female gametophyte; the egg cell and cium 1.75 mm). e Ovule with an MMC undergoing meiosis I; the polar nucleus are visible. Arc archespore, CC central cell, EC egg cell, nucleus of the MMC is at zygotene stage (gynoecium 2.5 mm). DMg degenerating megaspore, F funiculus, FG female gametophyte, f Developing ovule with a fully differentiated MMC (gynoecium FM functional megaspore, II inner integument, L1 L1 layer, MMC 3 mm); the outer integument is longer than the inner integument that megaspore mother cell, OI outer integument, PN polar nucleus. Scale covers approximately half of the nucellus. g Developing ovule at the bars a = 18 lm; b = 21 lm; c = 28 lm; d = 23 lm; e = 13 lm; end of megasporogenesis. The inner integument has resumed its f = 31 lm; g = 12 lm; h = 27 lm; i = 37 lm; j = 12 lm; growth; the functional megaspore (FM) is the meiotically derived cell k = 56 lm located at the chalazal pole, adjacent to three degenerating

(Fig. 2i). Antipodal cells are transiently evident at the have fused, forming a primary endosperm nucleus located chalazal pole of the embryo sac confirming formation of a adjacent to the egg apparatus (Fig. 2k). Polygonum-type embryo sac, but degrade soon after cellularization (Fig. 2j). The egg cell has a tear-like shape and Dynamics of pollen tube growth and double contains a conspicuous nucleus with prominent nucleolus fertilization usually located at its chalazal pole, contrary to the syn- ergids in which the nucleus is barely visible (Figs. 1u, 2j, We conducted manual cross-pollinations with flowers of k). At the onset of self-pollination, ovules contain fully IT97K, collecting and fixing gynoecia at consecutive time differentiated female gametophytes, and a voluminous, intervals following pollination, and observing pollen tube highly vacuolated central cell in which the two polar nuclei growth using aniline blue staining to establish the time of

123 172 Plant Reprod (2016) 29:165–177 fertilization. Typically, 50–70 % of flowers aborted gynoecium and the inner integument relative to the 24–48 h after pollination (HAP) without initiating viable megasporogenic stage, encompassing the full differenti- seed development. We estimated the distance from the ation of the MMC to the end of meiosis and differenti- surface of the stigma to the base of the funiculus of the ation of the functional megaspore (n = 296). During this most apical ovule to be 1.4 cm in length in cleared whole developmental timeframe, the minimum and maximum gynoecia. Pollen grains usually germinated within 3–4 length of a gynoecium was 0.5 and 4 mm, respectively. HAP following cross-pollination (Fig. 3a; Table 2). At 6 For the inner integument, we measured the maximum HAP, less than 10 % of germinated pollen tubes were number of longitudinal cell layers that were found at the within the apical region of the style, and by 12 HAP, the dorsal pole of the ovule; the maximum and minimum same proportion had reached the funiculus of the most number of cell layers score was 4 and 21, respectively. apical ovule, suggesting that rapid pollen tube growth The analysis of female meiotic configurations included occurs between 6 and 12 HAP (Fig. 3b; Table 2). Sperm stages encompassing megasporogenesis; in some cases, it cell delivery was observed within a degenerating synergid only included the MMC, and in others the four meioti- between 12 and 14 HAP. Although it was not possible to cally derived megaspores at different stages of devel- consistently visualize a degenerated synergid in whole- opment or degeneration (Fig. 4). In general, the meiotic mount cleared specimens, the sperm nucleus could be configuration width was poorly correlated with the three clearly identified within the primary endosperm nucleus of other parameters, suggesting that the volume of the the central cell, or in the cytoplasm or nucleus of the egg MMC and the megaspores along the dorsal–ventral axis cell (Fig. 3c–e). Following fertilization, the egg cell is highly variable. In contrast, the length of the meiotic acquires a spherical morphology reminiscent of zygotes in configuration was positively correlated with the sporo- many previously characterized legume species. phytic parameters; the best correlation (0.73) was obtained with the number of inner integument cells, Sporophytic parameters such as gynoecium length suggesting that the two developmental programs are and integument growth can be used to predict stages interrelated and at least partially synchronized. In addi- of megasporogenesis tion, the number of inner integument cells was also positively correlated with the length of the gynoecium, To assess the possibility of correlating sporophytic and confirming that the latter can be used as an approxima- gametophytic parameters during early ovule develop- tion to predict the developmental stage of the female ment, we measured morphological characteristics of the gametophyte.

Fig. 3 Dynamics of pollen tube growth and double fertilization in d Fertilization of the egg cell; a sperm cell (arrow) is adjacent to the cowpea. a Stigma showing pollen tube germination (arrows) 3 h after egg cell membrane (arrowhead). e Fertilization of the egg cell; a cross-pollination. b Pollen tube (arrow) having reached the micropyle sperm cell nucleus (arrow) is visible within the egg cell cytoplasm. of a fully differentiated ovule. c Fertilization of the central cell; a CC central cell, EC egg cell, PN polar nucleus. Scale bars sperm cell nucleus (arrow) is visible within the primary endosperm a = 40 lm; b = 50 lm; c–e = 20 lm polar nucleus; the egg cell has acquired a zygotic-like morphology. 123 Plant Reprod (2016) 29:165–177 173

Table 2 Dynamics of pollen tube growth in cross-pollinated ﬂowers of cowpea Time after pollination Observed Pollen tube Comments (h) styles length

1 15 Not germinated 3 15 50–100 lm The pollen tubes have germinated but have not penetrated the stigma 4 12 90–210 lm Less than 10 % of germinated pollen tubes penetrating the style 6 13 300–350 lm Less than 10 % of germinated pollen tubes penetrating the style 12 10 1.2–1.4 mm All ovules in the gynoecia showed the presence of a pollen tube at the surface of the funiculus

Fig. 4 Correlations between sporophytic and gametophytic parameters in ovules undergoing megasporogenesis. The length of the gynoecium, the number of inner integument cell layers (yellow dots in B), and the length and width of the meiotic conﬁguration were scored in developing ovules that were staged between the fully differentiated MMC and fully differentiated functional megaspore. DMg degenerating megaspore, FM functional megaspore, MMC megaspore mother cell, II inner integument, OI outer integument. Scale bars A = 10 lm; B = 20 lm. aThe distance measured for the meiotic conﬁguration length (red) and width (light blue)is highlighted in A and B

Immunolocalization of meiotic proteins in the ovule synaptonemal complex on the basis of an antibody raised against ASY1 (Fig. 5). The protocol is based on imple- Assessing any heterochronic divergence in developmental mented procedures used to observe structural features in traits related to such a highly dynamic process as megas- chromosomes of meiocytes of maize (Bass et al. 1997) and porogenesis requires novel tools to rapidly determine pro- the function of epigenetic pathways in Arabidopsis ovules tein localization in the ovule. Meiosis is of particular (Olmedo-Monﬁl et al. 2010). To adapt the protocol to the signiﬁcance for manipulation for the purpose of generating crassinucellate ovule of cowpea, we performed serial clonal seed. Therefore, we attempted the adaption in experiments using the same antibody that was used for cowpea of a whole-mount immunolocalization procedure Arabidopsis and maize, but increasing the time of exposure initially developed to follow the subcellular localization of to an enzymatic cocktail composed of driselase, pectolyase, proteins during female meiosis in Arabidopsis thaliana and cellulase. Whereas no signal was detected in ovules (Escobar-Guzman et al. 2015). In Arabidopsis, the proce- harboring an early archespore (data not shown), consistent dure can be used to follow meiotic proteins during ASY1 localization was restricted to the MMC at initial megasporogenesis, allowing the analysis of structural stages of megasporogenesis (Fig. 5). Initially localized details such as the formation of the axial element of the throughout the MMC nucleus, the signal was restricted to

123 174 Plant Reprod (2016) 29:165–177

Fig. 5 Whole-mount immunolocalization of ASY1 in the developing MMC. c Ovule of cowpea showing an MMC at prophase I of meiosis; ovule of Arabidopsis thaliana and cowpea IT86D. A polyclonal MMC chromatin is condensed at one of the edges of a conspicuous antibody against the maize protein ASY1 (green) was used in whole- nucleolus that presents a large nucleolar vacuole. L1 L1 layer, MMC mount immunolocalization; cells were counter-stained with propid- megaspore mother cell, II inner integument, OI outer integument. ium iodide (red). a Ovules of Arabidopsis showing ASY1 localization Scale bars a–c = 10 lm in the MMC. b Ovule of cowpea showing ASY1 localization in the one of the dorsal–ventral poles of the MMC nucleus at hybrid could be altered such that the offspring are identical prophase I. Counterstaining with propidium iodide showed to the parental hybrid plant, heterosis would be preserved that no signal was present in a large MMC nucleolus in harvested seeds. This could be achieved if gametes were containing a nucleolar vacuole. These results suggest that formed only by mitosis, viable endosperm was formed, and the signal indeed corresponds to the subcellular localiza- the embryo in the seed retained the hybrid genotype, tion of ASY1 at initial stages of megasporogenesis, raising mimicking apomixis, or asexual seed formation. We are the possibility of using the procedure to compare the attempting to develop self-reproducing hybrids in cowpea, dynamics of male and female meiosis in cowpea, and based, in part, on a recent combination of Arabidopsis examining protein localization at other stages of female mutants that have provided proof-of-concept of clonal seed reproductive development. formation (Marimuthu et al. 2011; Hand and Koltunow 2014; Gilbert 2015). The implementation of this type of technology requires new knowledge and experimental tools Discussion that depend on a detailed understanding of the genetic basis and molecular mechanisms that control reproductive The development and use of self-perpetuating improved development. hybrids of legume crops cultivated by smallholder farmers We have initiated a series of developmental studies that of sub-Saharan Africa and Southern Asia could substan- take advantage of cowpea local varieties to conduct a tially transform subsistence agriculture by affordably detailed cytological characterization of ovule and female offering superior germplasm and new sources of revenue gametophyte formation, identify correlation values on the basis of seeds adapted to local or regional condi- between some sporophytic and gametophytic parameters, tions. We hypothesize that if sexual reproduction of a and establish whole-mount immunocytological procedures

123 Plant Reprod (2016) 29:165–177 175 that allow a fast and reliable assessment of protein mitotic embryo sac formation and two rounds of mitosis expression in the ovule. As in other species of legume are observed (Ojehomon and Samyaolu 1970). This is investigated to date, the ovule of cowpea is anatropous, contrary to most Faboideae species in which the most bitegmic, and crassinucellate. But contrary to other legume chalazal megaspore undergoes three rounds of mitosis to species in which initial stages of primordium initiation are develop into a Polygonum-type of female gametophyte characterized by homogenous states of cell differentiation (Rembert 1969). Other exceptions include Trifolium repens (Mitchel 1975; Kennel and Horner 1985), the pre-meiotic and Vicia faba, in which an epichalazal megaspore ovule of cowpea is characterized by a cluster of up to 20 exceptionally can give rise to the functional megaspore differentiated L2 and L3 cells that are present immediately (Mitchel 1975; Rembert 1977), as well as Laburnum following the anticlinal divisions that give rise to the anagyroides and Pogamia, in which the Allium-type of incipient ovule bulge, but absent at later stages of elon- female gametogenesis was reported (Rembert 1966). Our gation, i.e., before differentiation of the archespore. results show that cowpea invariably forms a linear tetrad in Although the developmental nature of these cells remains which the surviving megaspore is located at the chalazal to be elucidated, their location and ephemeral clustered pole of the nucellus, indicating that the female gameto- organization suggests that they represent a proliferative phyte is indeed monosporic but of the Polygonum-type, stage of rapid cell divisions that give rise to the pre-meiotic which is the case for most species of the Faboideae primordium. reported to date. This observation was confirmed by the A single archespore differentiates in the ovule of cow- presence of three antipodals at the chalazal pole of the pea. As in some previously characterized legumes, but differentiating female gametophyte. At maturity, when the contrary to most angiosperms, the MMC is separated from egg apparatus is differentiated, the antipodals have the L1 by one additional sporophytic cell layer. Brown degenerated. (1917) was first to report this unusual position for the Our combination of cytological and morphometric female gamete precursor. In the case of P. vulgaris,a analyses has allowed the establishment of a female repro- careful analysis of cell divisions showed that the MMC ductive calendar that can be used to anticipate a range of origin was in the L2 layer, but that an unusual pattern of gametophytic stages on the basis of a sporophytic timeline divisions at the apex of the primordium gave rise to a that can be followed with help of specific parameters such second layer of cells separating the MMC from the L1 as bud size or gynoecium length. We propose that these (Brown 1917). Although not discussed by the authors, our parameters could be particularly useful to identify and own interpretation of data presented by Kennel and Horner estimate potential heterochronic events in comparison with (1985) indicates that the MMC in Glycine max is also wild-type gametophytic development in cowpea. Several separated from the L1 layer by one or two sporophytic cell studies have indicated that changes in cell specification and layers. In Vicia faba, Mitchel (1975) reports that a multi- method of reproduction caused by natural mechanisms, cellular archeosporium differentiates beneath two layers of such as diplospory or apospory, or by specific mutants nucellar cells. Contrary to P. vulgaris and cowpea, multiple affecting megasporogenesis can cause important differ- archespores differentiate in V. faba, as is also the case in V. ences in the timing of female meiosis or megaspore dif- americana, V. dasycarpa, and V. villosa (Rembert, 1969). ferentiation (Sharbel et al. 2010; Carman et al. 2011; In the case of V. faba as in G. max, the cytological char- Rodriguez-Leal et al. 2015). Although our analysis did not acterization is completely based on the analysis of sec- provide a significantly high positive correlation between tioned samples, complicating a possible determination of sporophytic and gametophytic parameters, the number of the patterns of cell division that occur in the L2 layer. cell layers present in the developing dorsal inner integu- Although a definitive elucidation of the MMC cell linage ment could provide a useful approximation. As is the case will require further cytological and genetic analysis, on the of most flowering species in which the correspondence basis of the classic study by Brown (1917) we propose the between male and female gametogenesis has been inves- hypothesis that the female gametic lineage in cowpea is of tigated, in cowpea the pollen grain differentiates more L2 origin, but that subsequent particularities of L2 layer rapidly than the female gametophyte. Ovule primordia divisions determine the final location of the MMC. Our harboring an archespore were present in flowers that con- results also suggest that the type of megagametogenesis tained anthers in which the pollen mother cell (PMC) was that prevails in cowpea should be reconsidered. In the case at the onset of meiosis; however, tetrads of microsporo- of cowpea, a single published report describing ovule and cytes were present in flowers in which the ovules contained female gametophyte development on the basis of thick a MMC at the onset of meiosis, indicating that male paraffin-embedded sections concluded that the variety meiosis occurs more rapidly than female meiosis. The ‘‘Adzuki’’ followed an Oenothera-type of megagametoge- slower progression of meiosis in the female was corrobo- nesis where the micropylar-most megaspore initiates rated by the presence of highly vacuolated uninucleate 123 176 Plant Reprod (2016) 29:165–177 microspores at stages in which the MMC had not yet Acknowledgments We thank T.J. Higgins for helpful comments on divided. At the onset of megagametogenesis, when the the manuscript and the International Institute of Tropical Agriculture for the provision of seed. This work was funded in part by a Bill and ovule exhibits a large functional megaspore, pollen grains Melinda Gates Foundation Grant to CSIRO (OP1076280). R.S.G. was contain a large vegetative cell and a small sperm cell, the recipient of a graduate scholarship from Consejo Nacional de confirming that the first mitotic division of microgameto- Ciencia y Tecnologia (CONACYT). N.S.L. was partially supported genesis occurs before the initiation of megagametogenesis. by the DuPont Pioneer regional initiatives to benefit local subsistence farmers. As expected, the fully differentiated pollen grain of cowpea contains one vegetative and two sperm cells before ger- Open Access This article is distributed under the terms of the mination. Despite the equivalency in the time of differen- Creative Commons Attribution 4.0 International License (http://crea tiation of the MMC and the PMC within a flower, the tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give developmental difference in timing is prevalent during both appropriate credit to the original author(s) and the source, provide a sporogenesis and gametogenesis, suggesting that male link to the Creative Commons license, and indicate if changes were meiosis occurs more rapidly than female meiosis. made. We also show that the adaptation of a previously published whole-mount immunolocalization procedure allows detection of proteins localized in single cells of the developing ovule, in particular the MMC. By contrast to References procedures that require sectioning or smearing to meio- Akibode S, Maredia M (2011) Global and regional trends in cytes from sporophytic cells, in cowpea the procedure production, trade and consumption of food legume crops. Report preserves the constitution of the ovule, providing a tem- submitted to CGIAR Special panel on impact assessment, 27 poral and spatial context to the meiotic division, and Mar 2011 opening the possibility for comparing the dynamics of Albertsen MC, Palmer RG (1979) A comparative light- and electron- microscopy study of microsporogenesis in male sterile (MS) and protein localization during female and male meiosis. We male fertile soybeans (Glycine max (L.) Merr.). ? 66:253–265 anticipate that the procedure will be useful for the analysis Andargie M, Pasquet RS, Gowda BS, Muluvi GM, Timko MP (2011) of developmental pathways related to the control of arch- Construction of a SSR-based genetic map and identification of espore differentiation, the characterization of mutants that QTLs for yield and domestication traits using recombinant inbred lines from a cross between wild X cultivated cowpea (V. affect megasporogenesis and require quantification of unguiculata (L.) Walp.). Mol Breed 28:413–420. doi:10.1007/ female meiotic defects within the same gynoecium, or the s11032-011-9598-2 observation of female meiosis in mutants that eventually Barrera-Figueroa B, Gao L, Diop NN, Wu Z, Ehlers JD, Roberts PA, lead to apospory or the formation of unreduced gametes. Close TJ, Zhu J, Liu R (2011) Identification and comparative analysis of drought-associated microRNAs in two cowpea The developmental timeline, gametogenic description, genotypes. BMC Plant Biol 11:127 and experimental tools presented here provide a necessary Bass HW, Marshall WF, Sedat JW, Agard DA, Cande WZ (1997) framework to support large-scale genomic efforts as a Telomeres cluster de novo before the initiation of synapsis: a source of information that could support marker-assisted three-dimensional spatial analysis of telomere positions before and during meiotic prophase. J Cell Sci 137:5–18 selection, laser-capture microdissection, transcriptional Bhaskaraiah KB, Shivashankar G, Virupakshappa K (1980) Hybrid profiling, traditional breeding, and genetic engineering for vigour in cowpea. Indian J Genet Plant Breed 40:334–337 the production of improved varieties of cowpea. Funda- Brown MM (1917) The development of the embryo-sac and embryo mental genome resources including large transcript data- in Phaseolus vulgaris. Bull Borrey Bot Club 44:535–546 Carman JG, Jamison M, Elliott E, Dwivedi KK, Naumova TN (2011) sets from specific cell types, genetic and physical maps, Apospory appears to accelerate onset of meiosis and sexual and genomic assemblies promise to establish the conver- embryo sac formation in sorghum ovules. BMC Plant Biol gent technological support necessary to develop improved 2011(11):9. doi:10.1186/1471-2229-11-9 hybrids. We propose that the framework of cytological Chehregani A, Tanaomi N (2010) Ovule ontogenesis and megagametophyte development in Onobrychis schahuensis Bornm. observations and experimental tools here described could (Fabaceae). Turk J Bot 34:241–248 also provide technical adaptations that could soon allow the Ehlers JD, Hall AD (1996) Genotypic classification of cowpea based analysis of reproductive alternatives as a first step to response to heat and photoperiod. Crop Sci 36:673–679 attempt the manipulation of sexual reproduction in cowpea Escobar-Guzman R, Rodriguez-Leal D, Vielle-Calzada JP, Ronceret A (2015) Whole-mount immunolocalization to study female for yield improvement. meiosis in Arabidopsis. Nat Protoc 10(10):1535–1542 Ghassempour H, Majd A, Assadi M, Gahremaninejad F, Nejadsatary Author contribution statement AMK and JPVC con- T, Semnani N, Moradi M (2011) Ovule ontogenesis and ceived and designed the research. RSG, SJ, and NSL megagametophyte development in Onobrychis sintenissii conducted the experiments, analyzed the data, and partic- Bormn. (Leguminosae-Papilionoideae). J Appl Biol Sci 6:51–54 ipated in manuscript preparation and writing. AMK and Gilbert N (2015) Gates foundation backs high-risk science for big wins. Nat Plants. doi:10.1038/nplants.2015.22 JPVC wrote the manuscript.

123 Plant Reprod (2016) 29:165–177 177

Hand ML, Koltunow AM (2014) The genetic control of apomixis: Popelka JC, Gollash S, Moore A, Molving L, Higgins TJ (2006) asexual seed formation. Genetics 197:441–450 Genetic transformation of cowpea (Vigna unguiculata L.) and Huang K, Mellor KE, Paul SN, Lawson MJ, Mackey AJ, Timko MP stable transmission of the transgenes to progeny. Plant Cell Rep (2012) Global changes in gene expression during compatible and 25(4):304–312 incompatible interactions of cowpea (Vigna unguiculata L.) with Pottorff M, Li G, Ehlers JD, Close TJ, Roberts PA (2014) Genetic the parasitic angiosperm Striga gesnerioides. BMC Genom mapping, synteny, and physical location of two loci for 13:402. doi:10.1186/1471-2164-13-402 Fusarium oxysporum f. sp. tracheiphilum race 4 resistance in Huynh B, Ehlers JD, Ndeve A, Wanamaker S, Lucas MR, Close TJ, cowpea [Vigna unguiculata (L.) Walp]. Mol Breed 33:779–791 Roberts PA (2015) Genetic mapping and legume synteny of Rembert DH Jr (1966) Megasporogenesis in Laburnum anagyroides. aphid resistance in African cowpea (Vigna unguiculata L. Medik. A case of bisporic development in Leguminosae. Trans Walp.) grown in California. Mol Breed 35:36 Ky Acad Sci 27:47–50 Kang YJ, Kim SK et al (2014) Genome sequence of mungbean and Rembert DH Jr (1969) Comparative megasporogenesis in Caesalpini- insights into evolution within Vigna species. Nat Commun. aceae. Bot Gaz 130:47–52 doi:10.1038/ncomms6443 Rembert DH Jr (1977) Ovule ontogeny, megasporogenesis, and early Kennel JC, Horner TH (1985) Megasporogenesis and Megagameto- gametogenesis in Trifolium repens (Papilionaceae). Am J Bot genesis in soybean, Glycine max. Am J Bot 72:1553–1564 64:483–488 Kumari S, Sahai G, Pandey SK, Pandey HC (2012) Identification of Rodriguez-Leal D, Leon-Martinez G, Vielle-Calzada J-Ph (2015) putative hybrids of cowpea (Vigna unguiculata (L) Walp.) and Natural genetic variation and its influence in the specification of their genetic evaluation using RAPD marker. Agric Sci Res J gametic precursors of Arabidopsis. Plant Cell 7(4):1034–1045 2:506–511 Rodriguez-Rianõs T, Valtuena FJ, Ortega-Olivencia A (2006) Lucas MR, Diop N, Wanamaker S, Ehlers JD, Roberts PA, Close TJ Megasporogenesis, megagametogenesis and Ontogeny of Aril (2011) SNP consensus mapping and comparative genomics of in Cytisus striatus and C. multiflorus (Leguminosae: Papil- cowpea [Vigna unguiculata (L. Walp.)]. Plant Genome ionoideae). Ann Bot 98:777–791 4:218–225 Sharbel TF, Voigt ML, Corral JM, Galla G, Kumlehn J, Klukas C, Lucas MR, Huynh B, Diop N, Roberts PA, Close TJ (2013) Markers Schreiber F, Vogel H, Rotter B (2010) Apomictic and sexual for breeding heat tolerant cowpea. Mol Breed 31:529–536 ovules of Boechera display heterochronic global gene expres- Lucas MR, Huynh B, Roberts PA, Close TJ (2015) Introgression of a sion. Plant Cell 22(3):655–671 rare haplotype from Southeastern Africa to breed California Singh BB (2014) Genetics and Breeding. In: Singh B (ed) Cowpea: blackeyes with larger seeds. Frontiers Plant Sci 6:126 the food legume of the 21st century. Crop Science Society of Lush WM, Evans LT (1981) Domestication and improvement of America, Madison cowpeas (Vigna unguiculata (L.) Walp). Euphytica 30:579–587 Soverna AF, Galati B, Hoc P (2003) Study of ovule and megaga- Marimuthu MP, Jolivet S, Ravi M, Pereira L, Davda JN, Cromer L, metophyte development in four species of subtribe phaseolinae Wang L, Nogue´ F, Chan SW, Siddiqi I, Mercier R (2011) (Leguminosae). Acta Biol Cracoviensia 45:63–73 Synthetic clonal reproduction through seeds. Science 331:876 Timko MP, Singh BB (2008) Cowpea, a multifunctional legume. In: Mitchel JP (1975) Megasporegenesis and microsporogenesis in Vicia Moore PH, Ming R (eds) Genomics of tropical crop plants. faba. Can J Bot 53:2804–2812 Springer, Berlin, pp 227–258 Moço MCC, Mariath JEA (2003) Ovule ontogeńesis and megasporo- Ushakumari R, Vairam N, Anandakumar CR, Malini N (2010) genesis in Adesmia latifolia (Spreng.) Vog. (Leguminosae- Studies on hybrid vigour and combining ability for seed yield Papilionoideae). Resvista Bras Bot 26:495–502 and contributing characters in cowpea (Vigna unguiculata). Ojehomon O, Samyaolu MO (1970) Ovule formation and embryo Electr J Plant Breed 1(4):940–947 development in persisting and abortive fruits of cowpea, Vigna Wien HC, Summerfield RJ (1980) Adaptation of cowpeas in West unguiculata (L.) Walp. Niger J Sci 4:31–40 Africa Effects of photoperiod and temperature response, in Olmedo-Monfil V, Durań-Figueroa N, Arteaga-Va´zquez M, Demesa- cultivars of diverse origin. In: Summerfield RJ, Bunting AH Are´valo E, Autran D, Grimanelli D, Slotkin K, Martienssen R, (eds) Advances in legume science. HMSO, London, pp 405–417 Vielle-Calzada J-Ph (2010) Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 464:628–632 Ortiz R (1998) Cowpeas from Nigeria: a silent food revolution. Outlook Agric 27(2):125–128

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